Reaction chip, reaction method, temperature controlling unit for gene treating apparatus and gene treating apparatus

ABSTRACT

The reaction chip of the present invention has a plurality of recesses  6  constituting a part of a reaction container and a groove constituting a part of a channel formed on at least one of one face of a first base material (resin base material  2 ) and one face of a second base material (metallic base material) and a notch  15  showing a gradual increase in width and a gradual increase in depth from one face  2   d  of the base material toward an inner wall surface  6   d  of the recess is formed on an edge of at least one recess in an extending direction of the groove. One face of the first base material and one face of the second base material are stuck together opposite to each other to form the plurality of reaction containers and the channel.

CROSS REFERENCE

This is a divisional of application Ser. No. 12/799,410, filed Apr. 22,2010, which is a continuation of PCT/JP2008/069273 filed on Oct. 23,2008, which claims priority to Japanese application numbers 2007-279107,2007-279108 and 2007-279109, which were filed on Oct. 26, 2007, all ofwhich are incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a reaction chip and a reaction methodsuitably used for a biochemical reaction such as a chemical reaction,DNA reaction, and protein reaction and a temperature controlling unitand a gene treating apparatus including the temperature controlling unitfor treatment such as amplification on genes contained in a biologicalsample.

2. Description of the Related Art

In recent years, in the field of, for example, a biochemical reactionsuch as a chemical reaction, DNA reaction, and protein reaction, atechnology called a μ-TAS (Total Analysis System) or Lab-on-Chip isstudied and put to practical use as a technique to treat a very smallquantity of sample solution on a chip. This enables a reactionexperiment, which required large-scale laboratory equipment and a largequantity of reaction reagents in the past, to perform with a smallquantity of reaction reagents using a reaction chip measuring several mmor less pre side.

Examples of this kind of biochemical reaction include a DNAamplification reaction by an enzyme reaction, hybridization reaction todetect a sequence of specimen DNA by using a probe DNA having a knownsequence, and detection reaction of SNP (monobasic polymorphism) in aDNA sequence. The invader (registered trademark) method and the TaqManPCR method are known as SNP detection methods (see, for example, PatentDocument 1).

When these reactions are caused using a chip, for example, to decide thesequence of a gene or DNA, a method by which a probe DNA is fixed ontoslide glass to allow a hybridization reaction thereon is known.

Further, a method by which a microscopic hole or dent called a well isformed on a chip to use the well as a reaction field is known.

A plurality of well-shaped reaction containers is mutually connected bya reagent solution channel installed from a reagent reservoir part (see,for example, Patent Document 2). When a reagent solution is fed by usingsuch a channel, it is important to fill a reaction container with thereagent solution to prevent bubbles from being left behind. If bubblesremain inside the reaction container, quantities and concentrations ofthe reagent solution in each reaction container fluctuate, leading tofluctuations of reaction states. Moreover, even if reaction states donot fluctuate, it is extremely probable that an error of photometricintensity is caused by the bubbles.

Thus, several methods are proposed to remove bubbles from the reactioncontainer.

As a method thereof, a liquid circuit having at least one channel insidea layered product formed by laminating a plurality of substrates inwhich a communication hole communicating the channel and outsidebypassing through at least one substrate is proposed (see, for example,Patent Document 3). Patent Document 3 discloses that the communicationhole is a hole formed in a single-crystal silicon substrate or glasssubstrate by using photolithography and has a tapered innercircumferential surface with an increasingly smaller opening area fromthe channel toward the outside to remove bubbles to the outside throughthe communication hole. Further, the communication hole preferably hasat least hydrophobicity and Patent Document 3 describes for this purposethat the substrate itself has hydrophobicity or adding hydrophobicity toa substrate having no hydrophobicity afterwards.

Further, a reaction chip having a channel passing through a firstsurface and a second surface and including a bubble trap to separatebubbles fed together with a sample in each sample hole is proposed (see,for example, Patent Document 4).

Incidentally, a reagent solution fed to a chip used for reactionanalysis is frequently a reagent solution having high viscosity such asorganic substance for a chemical reaction and extracted DNA, syntheticDNA, and enzyme for a biochemical reaction. When using such a reagentsolution, according to the method described in Patent Document 2 or 3,there is a possibility that bubbles are not sufficiently removed orseparated so that bubbles remain in the reaction container. Moreover,the method described in Patent Document 2 or 3 is a technologyapplicable only to a so-called open reaction container that is open tothe outer space and is not applicable to a closed reaction containerwhose outer circumference is completely enclosed by walls.

In addition, a method of making a reaction container hydrophobic withsurface treatment such as corona treatment and plasma treatment isfrequently used to remove bubbles. In this case, however, surfacemodification of the reaction container may occur to result in differentbatch reaction conditions such as a change in pH, posing a problem of apossibility of an intended desired reaction from being blocked.Moreover, if plasma treatment is applied, surface modification of thereaction container occurs, but the surface modification is hard topersist so that there is a problem that the state immediately aftertreatment cannot be maintained.

When a reaction is caused using these analysis chips, a reaction reagentis first arranged inside a plurality of well-shaped reaction containers.Next, a reaction reagent solution is fed to the plurality of well-shapedreaction containers via a channel by infusing the reaction reagentsolution into the analysis chip. Accordingly, the fixing reagent and thereaction reagent solution come into contact to start a reaction. Thewell-shaped reaction containers are heated during reaction if necessary.

However, according to the above reaction method, when the reactionreagent solution is fed into the well-shaped reaction container via thechannel, there is a possibility that the fixing reagent prearrangedinside the well-shaped reaction container flows out to adjacentwell-shaped reaction containers. Accordingly, there is a problem thatcontamination may be caused. There is also a possibility that the fixingreagent, reaction reagent solution, or fluorescent substance fordetection is diffused into the adjacent well-shaped reaction containersduring reaction in each well-shaped reaction container. Accordingly,there is a problem that it becomes impossible to measure accuratereaction data.

Thus, a reaction chip and a reaction method capable of preventing anoccurrence of such contamination and measuring accurate reaction dataare disclosed (see, for example, Patent Document 5).

The reaction chip described in Patent Document 5 is constituted by asubstrate forming a well-shaped reaction container and a cover materialcovering the substrate. The reaction method using the reaction chip isto cover a reaction reagent arranged in the well-shaped reactioncontainer with a hot-melt sealing compound and feed the reaction reagentsolution on top of the sealing compound before the sealing compoundbeing melted by heating to bring the reaction reagent and the reactionreagent solution into contact. According to the method, the reactionreagent will not flow out to adjacent well-shaped reaction containers,so that contamination can be prevented from occurring.

For a reaction chip used for biochemical reaction such as an enzymereaction, it is advantageous to use a substrate with a high thermalconductivity because such a reaction frequently requires heating areagent. However, if a reaction reagent is fixed onto a substrate with ahigh thermal conductivity, heat when the substrate and cover materialwere stuck together may be conducted to the reaction reagent, posing aproblem that activity of the reaction reagent is lowered or devitalized.Speaking of, for example, the reaction method described in PatentDocument 5, it is desirable to use a substrate with a high thermalconductivity as a substrate on the side on which a well-shaped reactioncontainer is formed, but in such a case, the above problem is caused.

If a reaction reagent is fixed onto a substrate with a high thermalconductivity and the reaction reagent is covered, like the method inPatent Document 5, with a hot-melt sealing compound, the sealingcompound is melted by heat when the substrate and cover material werestuck together, posing a problem that the sealing compound flows out toa channel of the reaction chip to block the channel or the shape of thesealing compound when re-solidified becomes unstable, leading toincomplete sealing of the reaction reagent. Because of this problem,there is a possibility that contamination cannot be sufficientlyprevented from occurring.

In these genetic tests, the amount of nucleic acid (DNA) contained in asample is amplified by the polymerase chain reaction (PCR) for the testand an attempt is being made to make the test faster by reducing thetime necessary for the PCR.

As a method of executing the PCR in a shorter time, an attempt is beingmade to execute the PCR with a smaller amount of sample and a reactioncontainer and a reaction apparatus (temperature controlling unit)therefor are devised.

Most reaction containers are made of synthetic resin that does notinhibit a biological reaction and a reaction is caused by reducing areaction volume to several tens microliter. In addition, in someinstances, the reaction container is formed from aluminum.

In such reaction containers, a PCR reaction is allowed to occur withouta minimum amount of sample being evaporated by a heating unit beingbrought into contact from above and below by a reaction apparatusdescribed in Patent Document 6 or 7.

The reaction apparatus described in Patent Document 6 or 7 causes no bigproblem if the reaction container is formed from a single material.However, if a PCR reaction is allowed to occur by using a containerconstructed by separate materials having different thermalconductivities in an upper part and a lower part of the reactioncontainer for the purpose of improving performance of the reactioncontainer, the temperature distribution of the sample inside thereaction container becomes inhomogeneous due to a difference in thermalconductivity, posing a problem that the PCR reaction does not proceedsmoothly.

-   Patent Document 1: Japanese Patent Application Laid-Open No.    2002-300894-   Patent Document 2: Japanese Patent Application National Publication    No. 2002-503336-   Patent Document 3: Japanese Patent Application Laid-Open No.    9-257748-   Patent Document 4: Japanese Patent No. 2955229-   Patent Document 5: Japanese Patent Application Laid-Open No.    2007-090290-   Patent Document 6: Japanese Patent No. 3661112-   Patent Document 7: Japanese Patent No. 3686917

SUMMARY OF THE INVENTION

The present invention has been made to solve the above problems.

A first object of the present invention is to provide a reaction chipcapable of easily feeding a liquid without surface modification of areaction container and leaving bubbles behind and performing accuratedetection and measurement of a desired reaction.

A second object of the present invention is to provide a reaction methodcapable of reliably preventing an occurrence of contamination andmeasuring accurate reaction data without lowering or devitalizingactivity of a reaction reagent.

A third object of the present invention is to provide a temperaturecontrolling unit for a gene treating apparatus and a gene treatingapparatus capable of treating genes contained in a gene sample filled ina reaction container swiftly and appropriately even if the reactioncontainer constructed from a plurality of materials having differentthermal conductivities is used.

A thorough examination by the present inventors to attain the firstobject shows, as a result, that a conventional reaction containercomposed of a recess having a steep inner wall surface traps bubblesparticularly in a space sandwiched between the inner wall surfacepresent in a flow direction of a reagent solution and a bottom.Therefore, the present inventors conceive the constitution of thepresent invention by realizing that it becomes harder for bubbles toremain in a recess by making such an inner wall surface smaller andadopting a configuration that allows a reagent solution to flow smoothlynear the recess.

A reaction chip in the present invention is a reaction chip having aplurality of reaction containers constituted by a pair of base materialsto cause a reaction between a reagent and a reagent solution and achannel that mutually communicates the plurality of reaction containersto feed the reagent solution to the plurality of reaction containers,including forming a plurality of recesses constituting a part of thereaction container on at least one of one face of a first base materialand one face of a second base material of the pair of base materials,forming a groove constituting a part of the channel at a positioncorresponding to between the recess and the recess on at least one ofone face of the first base material and one face of the second basematerial, forming a notch showing a gradual increase in width and agradual increase in depth from one face of the base material where therecess is formed toward an inner wall surface of the recess on an edgeof at least one recess of the recesses in an extending direction of thegroove, and forming the plurality of reaction containers and the channelby one face of the first base material and one face of the second basematerial being stuck together facing each other.

In the reaction chip of the present invention, an angle formed by oneface of the base material where the recess is formed and the inner wallsurface of the notch is smaller than the angle formed by one face of thebase material where the recess is formed and the inner wall surface ofthe recess.

In the reaction chip of the present invention, the notch is formed on atleast an inflow side of the reagent solution flowing through the grooveon the edge of the recess in the extending direction of the groove.

In the reaction chip of the present invention, a configuration may beadopted in which the notch is formed on at least an outflow side of thereagent solution flowing through the groove on the edge of the recess inthe extending direction of the groove so that the notch formed on theinflow side of the reagent solution and the notch formed on the outflowside of the reagent solution form a line symmetric shape.

In the reaction chip of the present invention, the recess has a columnarspace having the inner wall surface at substantially right angles to oneface of the base material where the recess is formed on at least anopening side, and the maximum depth on the edge of the notch isshallower than the depth of the columnar space.

An outer shape of the recess is circular in plane view and a plane shapeof the notch is defined, when two tangents to a circle forming an outeredge of the recess are drawn from one point on one face of the basematerial where the recess is formed in the extending direction of thegroove, by an area inside the two tangents.

In the reaction chip of the present invention, a center line in theextending direction of the groove of the notch is aligned with thecenter line of the groove on a same straight line.

To achieve the second object, a reaction method of the present inventionis a reaction method using a reaction chip having a channel thatmutually communicates a plurality of reaction containers constituted bya pair of base materials and the plurality of reaction containers,including the steps of arranging a reagent inside a recess by using, ofthe pair of base materials, the first base material having the recessconstituting a part of the reaction container formed therein, sealingthe reagent with a hot-melt sealing compound, producing the reactionchip having the reaction containers in which the reagent is arranged andthe channel by sticking the second base material constituted by amaterial whose thermal conductivity is higher than that of the firstbase material and the first base material together, feeding a reagentsolution into the reaction containers through the channel, and causing areaction of the reagent and the reagent solution to proceed while heatbeing added from a side of the second base material after the reagentand the reagent solution being brought into contact by heating thereaction chip from the side of the second base material to melt thesealing compound.

In the reaction method of the present invention, the first base materialand the second base material are stuck together through thermal weldingof a sealant layer provided on at least one side of the first basematerial and the second base material by adding heat from the side ofthe second base material.

In the reaction method of the present invention, the recesscorresponding to the recess in the first base material is also formed inthe second base material to constitute the reaction container by boththe recess of the first base material and the recess of the second basematerial.

In the reaction method of the present invention, a resin material isused as the first base material and a metallic material is used as thesecond base material.

In the reaction method of the present invention, the sealing compound isconstituted by a material that is insoluble in neither the reactionreagent nor the reagent solution.

In the reaction method of the present invention, the reaction containeris a reaction container for enzyme reaction.

To achieve the third object, a temperature controlling unit for genetreating apparatus of the present invention is a temperature controllingunit for gene treating apparatus that treats a gene inside a gene sampleby heating/cooling the gene sample filled in a reaction containercomposed of a first member arranged in an upper part and a second memberhaving a different thermal conductivity from that of the first memberand arranged in a lower part, including a first temperature controllingunit arranged in such a way to allow contact with a top face of thereaction container, a second temperature controlling unit arranged insuch a way to allow contact with an undersurface of the reactioncontainer and also arranged in such a way to be able to sandwich thereaction container between the first temperature controlling unit andthe second temperature controlling unit, a pair of metallic platesarranged on surfaces where the first temperature controlling unit or thesecond temperature controlling unit is in contact with the reactioncontainer, a pair of heat conduction members arranged on surfaces of thepair of metallic plates facing the reaction container and also arrangedin such a way to allow contact with the top face and the undersurface ofthe reaction container, a first heat dissipation unit provided incontact with the first temperature controlling unit, and a second heatdissipation unit provided in contact with the second temperaturecontrolling unit.

According to a temperature controlling unit for gene treating apparatusof the present invention, heat of the first temperature controlling unitor the second temperature controlling unit is dissipated uniformly by apair of metallic plates and transmitted efficiently to a reactioncontainer by a pair of heat conduction members.

The temperature controlling unit for gene treating apparatus of thepresent invention further includes a control unit connected to the firsttemperature controlling unit and the second temperature controlling unitto control temperatures of the first temperature controlling unit andthe second temperature controlling unit, wherein the control unit mayexercise temperature control of the first temperature controlling unitand the second temperature controlling unit independently based on thethermal conductivities of the first member and the second member.

In this case, the temperature difference of a gene sample between thefirst member and the second member becomes smaller, so that the gene canbe treated more suitably.

A gene treating apparatus of the present invention includes atemperature controlling unit for gene treating apparatus of the presentinvention.

According to a gene treating apparatus of the present invention, a genecan be treated quickly and suitably even if a reaction containercomposed of a first member and a second member is used.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing (s) will be provided by the Office upon request andpayment of the necessary fee.

FIG. 1 is a perspective view of a reaction chip according to anembodiment of the present invention.

FIG. 2 is a plan view of a resin base material constituting the reactionchip.

FIG. 3 is a plan view of a metallic base material constituting thereaction chip.

FIG. 4 is an enlarged view of a recess of the resin base material, andFIG. 4A is a perspective view thereof, FIG. 4B is a plan view thereof,and FIG. 4C is a side sectional view thereof.

FIG. 5 is a diagram showing another example of the recess, and

FIG. 5A is a perspective view thereof and FIG. 5B is a side sectionalview thereof.

FIGS. 6A, 6B and 6C are process sectional views showing a reactiondetection method using the reaction chip following procedures thereof.

FIGS. 7A, 7B and 7C are process sectional views when the reaction chipof another type is used.

FIG. 8 is a perspective view of a reaction chip in an embodiment of thepresent invention.

FIG. 9A is a plan view of the reaction chip and FIG. 9B is a sectionalview along a line A-A′ in FIG. 8.

FIGS. 10A, 10B and 10C are process sectional views showing a reactionmethod using the reaction chip following procedures thereof.

FIG. 11 is a sectional view showing another example of the reactionchip.

FIG. 12 is a perspective view showing the configuration of a geneamplifying apparatus including a temperature controlling unit for geneamplifying apparatus according to an embodiment of the presentinvention.

FIG. 13 is a schematic sectional view showing a state where a reactioncontainer is sandwiched by the temperature controlling unit for geneamplifying apparatus.

FIG. 14 is an enlarged sectional view in the vicinity of the reactioncontainer in FIG. 13.

FIG. 15 is a perspective view showing the reaction container.

FIG. 16 is a sectional view along a line A-A′ in FIG. 15.

FIG. 17 is a sectional view along a line B-B′ in FIG. 15.

FIG. 18 is a graph showing a temperature cycle by the PCR method,temperature control by the temperature controlling unit for geneamplifying apparatus, and temperature changes of each unit of thereaction container.

FIG. 19 is a graph showing temperature changes when the reactioncontainer 100 is used in a conventional gene amplifying apparatus.

FIG. 20 is an enlarged view of the recess of the reaction chip inComparative Example 1, and FIG. 20A is a plan view thereof and FIG. 20Bis a side sectional view thereof.

FIGS. 21A, 21B and 21C are process sectional views when the reactionchip of Comparative Example 1 is used.

FIG. 22 is a photo shooting conditions inside the reaction container ofthe reaction chip of Example 1.

FIG. 23 is a photo shooting conditions inside the reaction container ofthe reaction chip of Comparative Example 1.

FIG. 24A is a plan view showing reagent arrangement of Example 2 andComparative Example 2 of the present invention, FIG. 24B is a sectionalview of the reaction chip of Example 2, and FIG. 24C is a sectional viewof the reaction chip of Comparative Example 2.

FIGS. 25A and 25B are graphs showing reaction results of Example 2.

FIG. 26 is a graph showing reaction results of Comparative Example 2.

1: Reaction chip, 2: Resin base material (first base material), 3:Metallic base material (second base material), 4: Reaction container, 5:Channel, 6: Recess (of a resin base material), 6 a: Columnar space, 6 b:Truncated cone shaped space, 11: Recess (of a metallic basematerial),12: Groove, 15, 16: Notch, 21: Reaction chip, 22: Cover material (firstbase material), 23: Substrate (second base material), 24: Reactioncontainer, 25: Channel, 26: Recess (of a cover material), 27: Recess (ofa substrate), 28: Groove, 30: Reagent solution injecting hole, 31:Through hole, 41: Temperature controlling unit for gene amplifyingapparatus, 42: Gene amplifying apparatus (gene treating apparatus), 43:Movable carriage, 44: Measuring unit, 45: Moving unit, 46: Rail, 47:Emission detection unit, 48: Measuring unit moving unit, 49: First unit,50: Second unit, 51: Support arm, 52: First temperature controllingunit, 53: First heat sink (first heat dissipation unit), 54: Secondtemperature controlling unit, 55: Second heat sink (second heatdissipation unit), 56: First heat conduction layer, 58: Control unit,59: Metallic plate, 60: Second heat conduction layer (heat conductionmember), 61: Heat insulating material, 100: Reaction container, 101:First member, 102: Second member, 103: Well, 104: Reagent, 105: Channel,106: Injecting hole, 107: Deaeration port, S: Reagent, W: Sealingcompound, L: Reagent solution

DETAILED DESCRIPTION OF THE INVENTION First Embodiment

An embodiment of the present invention will be described below withreference to FIGS. 1 to 7.

The present embodiment shows an example of a reaction chip forbiochemical reaction analysis.

FIG. 1 is a perspective view of a reaction chip in the presentembodiment. FIG. 2 is a plan view of a resin base material (first basematerial) constituting the reaction chip. FIG. 3 is a plan view of ametallic base material (second base material) constituting the reactionchip. FIGS. 4A to 4C are enlarged views of a recess of the resin basematerial and FIG. 4A is a perspective view thereof, FIG. 4B is a planview thereof, and FIG. 4C is a side sectional view thereof. FIGS. 5A and5B are diagrams showing another example of the recess and FIG. 5A is aperspective view thereof and FIG. 5B is aside sectional view thereof.FIGS. 6A to 6C are process sectional views showing a reaction detectionmethod using the reaction chip following procedures thereof. FIG. 7 is aprocess sectional view when the reaction chip of another type is used.

For convenience of description, it is assumed below that the side ofresin base material positioned on the upper side, when a fluorescentreaction is detected or measured, is the “upper side” and the side ofmetallic base material positioned on the lower side is the “lower side”.

A reaction chip 1 in the present embodiment is a small chip that has, asshown in FIG. 1, a rectangular shape whose plane shape has about severaltens mm both in length and width and a thickness of about several mm.The reaction chip 1 is constituted by a resin base material 2 (firstbase material) and a metallic base material 3 (second base material)arranged on the lower side of the resin base material 2. In the reactionchip 1 in the present embodiment, the resin base material 2 has recessesconstituting reaction containers 4 formed therein and the metallic basematerial 3 has recesses constituting the reaction containers 4 andgrooves constituting channels 5 formed therein.

A plate material of polypropylene superior in terms of lighttransmission, heat resistance, chemical resistance, molding workability,and strength may be used as the resin base material 2. In addition tothis, a resin material such as polycarbonate, acryl(polymethylmethacrylate), polyethylene terephthalate, polyethylene, polyvinylchloride, and polystyrene as materials having similar characteristics.

The thickness of the resin base material 2 is preferably such that theresin base material 2 should not easily be bent while being used.Moreover, the resin base material 2 may be formed by two types of resinor more being bonded. In such a case, various base materials inaccordance with physical properties of the reaction reagent or samplecan be produced by preparing base materials making the most ofcharacteristics of each resin so that different base materials can beused for different purposes. For example, the material for the upperpart and that for the lower part of the base material may be separated.Further, the material of base material is not limited to resin andquartz glass may also be used.

The resin base material 2 has, as shown in FIG. 2, a plurality (in thepresent embodiment 36 recesses, 6 rows×6 columns) of recesses 6constituting a part of the reaction container 4 formed on theundersurface thereof. These recesses 6 do not mutually communicate andthus is isolated. The plane shape of the recess 6 is circular and thesectional shape thereof is, as shown in FIG. 4C, a columnar space 6 a onthe side closer to the undersurface of the resin base material 2 and atruncated cone shaped space 6 b on the side farther from theundersurface. The shape of the recess 6 (together with the plane shapeand sectional shape) can appropriately be designed such that the wholereagent can reliably be accommodated at the bottom of the recess 6 inaccordance with the amount of reagents or reagent solutions necessaryfor a reaction. However, if, like the present embodiment, aconfiguration in which the recess has a truncated cone shaped space onthe bottom side of a columnar space is adopted, a liquid settles down ina portion of the truncated cone shaped space with stability so that areagent or a fixing agent can reliably be accommodated. Particularly, ifthe base material on the side on which a recess is formed is constitutedby a material having light transmission, the flat bottom of thetruncated cone shaped space is suitable for detection of a fluorescentreaction so that fluorescence can be detected accurately.

The recess 6 is formed by methods of cutting a resin plate constitutingthe resin base material 2, injection-molding a resin materialconstituting the base material or the like. In view of miniaturizationof the reaction chip, the diameter of the recess 6 (the reactioncontainer 4) is preferably about 0.01 mm or more and 10 mm or less. Thismakes feeding of a reagent solution described later relatively easierand can reduce the amount of a fixing reagent or reagent solution to aminimum. As described later, a reagent necessary for an incipientreaction of a sample containing DNA added when used is arranged in eachof the recesses 6 of the resin base material 2. Alternatively, thereagent may be arranged only in a portion of the recesses 6 so thatplural types of reactions can be caused in one reaction chip.

The configuration of the recess 6 will be described later.

As shown in FIGS. 1 and 2, a plurality (in the present embodiment, six)of reagent solution injecting holes 7 is provided at one end on the topface (the surface on the opposite side of the surface where the recesses6 are formed) of the resin base material 2. The reagent solutioninjecting hole 7 is communicatively connected to a through hole (notshown) passing through a top plate part 2 a of the resin base material 2and is formed in a cylindrical shape protruding upward. Air dischargeholes 8 are provided at the other end of the resin base material 2 onthe opposite side of the side where the reagent solution injecting holes7 are provided. The air discharge hole 8 has a cylindrical shape, has athrough hole in the center, and has a filter (not shown) filled in thethrough hole. The filter has a function to smoothly pass a reagentsolution by allowing air to pass through while the reagent solutionflows. On the other hand, when the reagent solution that has flownthrough the channel reaches the air discharge hole 8, the filter has afunction to prevent the reagent solution from flowing out by holdingback the reagent solution. A frame part 2 b that hangs down from the topplate part 2 a is provided on the edge of the top plate part 2 a of theresin base material 2, and the metallic base material 3 is arranged andfixed inside the frame part 2 b.

An aluminum sheet, for example, can be used as the metallic basematerial 3 and a resin sealant layer (not shown) is formed on one sideof the aluminum sheet. The resin sealant layer is made of polypropyleneas a main material and is a bonding layer that can thermally be weldedwith the metallic base material 3 and the resin base material 2.

In addition to aluminum, copper, silver, nickel, brass, or gold may beused as the material of the metallic base material 3.

The metallic base material 3 has, as shown in FIG. 3, a plurality (inthe present embodiment, 36) of recesses 11 constituting a part of thereaction container 4 formed on the top face of the metallic basematerial 3. These recesses 11 are formed at a position corresponding tothe recesses 6 of the resin base material 2 when the metallic basematerial 3 and the resin base material 2 are aligned.

In contrast to the recess 6 of the resin base material 2, the sectionalshape of the recess 11 has, as shown in FIG. 6, a substantiallyhemispheric shape. While the recesses 6 and 11 correspond one-to-onebetween the resin base material 2 and the metallic base material 3 inthe present embodiment, one-to-one correspondence may not necessarily berealized or recesses of different sizes may be formed depending onpurposes of use. In the present embodiment, the volume of the recess 6and that of the recess 11 are substantially the same on the resin basematerial 2 side and the metallic base material 3 side.

Moreover, a groove 12 constituting a part of the channel 5 is formedbetween the recesses 11 on the top face of the metallic base material 3.The reaction chip 1 in the present embodiment has, as shown in FIGS. 1and 3, six sets of the channel 5 and the six recesses 11 (reactioncontainers 4) are serially connected in one set of the channel 5. Aslight recess 13 is formed at a position corresponding to each of thereagent solution injecting holes 7 and each of the air discharge holes8, and the groove 12 is also formed between the recess 13 and the recess11. Thus, a reagent solution injected from each of the reagent solutioninjecting holes 7 flows through the channel 5 and, after the sixreaction containers 4 being filled successively, is held back by thefilter of the air discharge holes 8.

The configuration of the recess 6 of the resin base material 2 will bedescribed in detail below using FIGS. 4A to 4C. Incidentally, FIG. 4Aalone is depicted by inverting vertically so that the shape of therecess 6 can be made easier to view.

The recess 6 has a circular shape with a diameter T1 as a plane shape,the columnar space 6 a with the diameter T1 and a depth D1 as asectional shape on the side closer to the undersurface of the resin basematerial 2, and the truncated cone shaped space 6 b with a diameter T2of the circle at the bottom and a depth D2 as a sectional shape on theside farther from the undersurface (bottom side of the recess). A notch15 showing a gradual increase in width and a gradual increase in depthfrom an undersurface 2 d of the resin base material 2 toward an innerwall surface 6 d of the recess 6 is formed on edges of both an inflowside and an outflow side of a reagent of the recesses 6 along anextending direction of the groove 12 (the channel 5). The notch 15 onthe inflow side and that on the outflow side of a reagent have the sameshape and are arranged, as shown in FIG. 4B, symmetrically with respectto a center line C extending in a direction perpendicular to theextending direction of the groove 12. The notch 15 on the inflow sideand that on the outflow side of a reagent may have different shapes andmay not necessarily be symmetric with respect to the center line C.

As shown in FIG. 4A, the notch 15 has a shape cut out like a triangularpyramid shape from the undersurface 2 d of the resin base material 2toward the inner wall surface 6 d of the recess 6 in the extendingdirection of the groove 12. Therefore, the bottom of the notch 15 formsan acute valley line 15 b. In the columnar space 6 a of the recess 6,the inner wall surface 6 d rises steeply at substantially right anglesto the undersurface 2 d of the resin base material 2 and, as shown inFIG. 4C, an angle θ1 formed by the undersurface 2 d of the resin basematerial 2 and the valley line 15 b of the notch 15 becomes sufficientlysmaller than an angle θ2 (θ2≈90° formed by the undersurface 2 d of theresin base material 2 and the inner wall surface 6 d of the recess 6 dueto formation of the notch 15. The bottom of the notch 15 need notnecessarily be an acute valley line and may be, for example, a gentlycurved surface.

The plane shape of the notch 15 is defined, when two tangents L1 and L2to a circle forming an outer edge of the recess 6 are drawn from anypoint A on the undersurface 2 d of the resin base material 2 in theextending direction of the groove 12, as shown in FIG. 4B, by aninternal area enclosed by the two tangents L1 and L2 and the circle. Ifthe angle formed with the two tangents L1 and L2 is θ, θ is preferably5° or more. This is because θ of 5° or less makes processing harder andalso an effect of bubble removal is hardly achieved. A distance T3 fromthe point A to an intersection of the valley line 15 b of the notch 15and the circle can be appropriately decided in accordance with aninterval between the adjacent recesses 6 of the resin base material 2.The recess 6 is designed such that the center line (the valley line 15b) along the extending direction of the groove 12 of the notch 15 isaligned with the center line of the groove 12 of the metallic basematerial 3 on the same straight line. With the above plane shape, theplane shape of each of the recesses 6 in the present embodiment has ashape that has sufficiently small flow resistance so that a reagentsolution flows extremely smoothly.

On the other hand, the sectional shape when the notch 15 is cut alongthe center line extending in the extending direction of the groove 12 ofthe notch 15 is as shown in FIG. 4C. Lines represented as solid linesare what visually appears of the notch 15 and those represented as chaindouble-dashed lines are a (virtual) triangle for design when the recess6 is designed so that the notch 15 is cut out in a triangular shape asthe sectional shape. The virtual depth at a vertex positioned inside thecolumnar space 6 a of the triangle is set as D3. Consequently, the pointwhere the valley line 15 b of the notch 15 and the inner wall surface 6d of the recess 6 (the columnar space 6 a) intersect is a point wherethe distance of the intersection point from the undersurface 2 d of theresin base material 2 becomes a maximum depth D4 of the notch 15.

Examples of dimensions in the present embodiment are: the diameter T1 ofthe recess (columnar space) is 3 mm, the diameter T2 of the truncatedcone shaped space is 2 mm, the distance T3 from the point A to anintersection of the valley line 15 b of the notch 15 and the circle is 1mm, the depth D1 of the columnar space 6 a is 0.8 mm, the depth D2 ofthe truncated cone shaped space 6 b is 0.7 mm, and the virtual depth D3at a vertex positioned inside the columnar space 6 a of the virtualtriangle is 0.6 mm. These dimensions are only examples and the designcan be changed when appropriately.

In the example shown in FIG. 4C, the depth D4 to be the maximum depth ofthe notch 15 is shallower than the depth D1 of the columnar space 6 a ofthe recess 6. Thus, even if the notch 15 is formed on the edge of thecolumnar space 6 a, the vertical inner wall surface 6 d in the columnarspace 6 a of the recess 6 remains at the position of the valley line 15b of the notch 15. Therefore, in this example, a volume capable ofaccommodating reagents or wax described later can be ensured and alsoreagents or wax can reliably be accommodated inside the columnar space 6a and the truncated cone shaped space 6 b so that the top face of thewax is positioned in a part of the columnar space 6 a in which thevertical inner wall surface 6 d remains.

Alternatively, as shown in FIGS. 5A and 5B, a design in which adimension D4′ to be the maximum depth of a notch 16 is made equal to thedepth D1 of the columnar space 6 a of the recess 6 may be adopted. Inthis case, the size of the notch 16 becomes substantially larger thanthe configuration shown in FIG. 4B and no vertical inner wall surface inthe columnar space 6 a of the recess 6 remains at the position of avalley line 16 b of the notch 16. In this example, while the volume ofthe space to accommodate reagents or wax is slightly reduced whencompared with the configuration shown in FIG. 4B, reagents are madeeasier to flow, so that bubbles can be prevented from remaining.

A manufacturing method of a reaction chip in the present embodiment willbe described below using FIGS. 6 and 7.

As shown in FIG. 6A, after a resin sealant layer is formed on one sideof an aluminum sheet to produce a base material sheet, the metallic basematerial 3 including a plurality of recesses 11 and a plurality ofgrooves 12 is produced by a method of drawing on the base material sheetor the like. On the other hand, the resin base material 2 having aplurality of recesses 6 is formed by a method of injection molding orthe like. Then, the notch 15 is formed on the edge of the recess 6 by amethod of cutting or the like. Any cutting method can be selected. Theresin base material 2 including the plurality of recesses 6 having thenotch 15 from the start may be produced by the method of injectionmolding or the like.

Next, the opening of the recess 6 is directed upward and a reagent S isput into the plurality of recesses 6 of the resin base material 2 beforebeing fixed. Further, the reagent S is covered with wax W before beingsolidified. The wax (fixing material) herein is a material that coversthe reagent arranged inside the recess 6 and remains in a solid stateuntil a reaction between a reagent and a reagent solution begins. Thewax may be a single material or made up of a plurality of materials (forexample, a mixture). In embodiments of the present embodiment, hot-meltwax is used, but wax that is melted by a factor other than heat orbreaks so that a reagent and a reagent solution mix may be adopted. Anykind of wax that does not prevent a reaction between a reagent and areagent solution but melts at a necessary temperature may be selected.

The reagent used in the present invention may be in a solid state or aliquid state.

Next, as shown in FIG. 6B, the resin base material 2 to which thereagent S is fixed is placed on top of the metallic base material 3 suchthat surfaces on which the mutual recesses 6 and 11 are formed face eachother and then heat is added. The resin sealant layer on the surface ofthe metallic base material 3 melts and the resin base material 2 and themetallic base material 3 are welded. With the above processes, areaction chip including a plurality of reaction containers 4 and aplurality of channels 5 is completed.

The method of thermal welding can be appropriately selected from methodssuch as the heat sealer, laser welding, and ultrasonic welding.Alternatively, instead of welding, the resin base material 2 and themetallic base material 3 may be stuck together. In such a case, anadhesive used for pasting can appropriately be selected from anyadhesive on the market that does not inhibit the target reaction.Alternatively, a method of mechanical crimp by a roller or the like withinvolvement of an adhesive material between the resin base material 2and the metallic base material 3 may be adopted.

Next, as shown in FIG. 6C, a reagent solution L is fed into each of thereaction containers 4 of the completed reaction chip. After the reagentsolution L being fed, the channel 5 is blocked by plastically deforminga part of the groove 12 of the metallic base material 3 to isolate eachof the reaction containers 4. By isolating each of the reactioncontainers 4, mixing of unnecessary reagents between the adjacentreaction containers 4 can be prevented. As a means for plasticdeformation of the groove 12 of the metallic base material 3, anexternal force may mechanically be applied to a part of the groove 12from outside by using a device, or an external force may be applied byhand. Then, when the temperature of the reaction chip 1 is controlled toa predetermined temperature (the melting point of the wax W or higher),the solidified wax W is melted and the reagent S and the reagentsolution L are mixed inside the reaction container 4, initiating areaction. In the present embodiment, the resin base material 2 made ofpolypropylene has greater transparency, so that fluorescence duringreaction can be detected from outside on the side of the resin basematerial 2.

In the foregoing, the manufacturing method of a reaction chip in thepresent embodiment has been described by using an example of a reactionchip having the recess 11 and the groove 12 formed on the side of themetallic base material 3, but a configuration in which the recesses andgrooves are all formed on the side of the resin base material and a flatplate material or a film 3A (a material other than metal is alsoallowed) is used on the side of the metallic base material to cover therecesses of the resin base material with the flat plate material or thefilm 3A may be adopted. An example thereof is shown in FIGS. 7A to 7C.This example is different from the above example only in which basematerial to form the recesses and grooves and the basic manufacturingprocesses are the same and thus, the same reference numerals areattached to components in FIGS. 7A to 7C that are common to those inFIGS. 6A to 6C and a description thereof is omitted.

The reaction chip 1 in the present embodiment has the notch 15 formed onthe edges on the inflow side and outflow side of the reagent L of therecesses 6 formed on the resin base material 2 and thus, the reagentsolution L flows smoothly near the recess 6 so that the inflow of thereagent solution L from the channel 5 into the reaction container 4 andthe outflow of the reagent solution L from the reaction container 4 tothe channel 5 become smooth. Thus, even if the reagent solution Lcontaining bubbles flows in, bubbles pass through the reaction container4, so that the frequency of bubbles remaining the recess 6 cansignificantly be decreased. As a result, the desired reaction canaccurately be detected or measured by using the reaction chip 1 in thepresent embodiment. Moreover, there is no need ofhydrophobic/hydrophilic treatment and surface treatment such as coronatreatment and plasma treatment because bubbles can be removed frominside the recess 6 only by forming the notch 15 on the edge of therecess 6.

The technical scope of the present invention is not limited to the aboveembodiment and various modifications can be made without deviating fromthe spirit of the present invention. For example, while groovesconstituting a channel are formed only in the metallic base material inthe above embodiment, grooves may also be formed in the resin basematerial in accordance with the volume of a reagent solution so that thechannel is constituted by both the metallic base material and resin basematerial. Further, an example in which the reaction contains have allthe same size is described in the above embodiment, but instead thereof,a plurality of reaction containers having different sizes may beincluded. In this case, the shape or dimensions of the notch may beoptimized by adjusting to the size of each reaction container. Moreover,concrete configurations such as the shape, number, and arrangement ofthe reaction containers and channel, materials and dimensions of eachbase material, various methods used in each manufacturing processexemplified in the above embodiment are only examples and may be changedwhen appropriate.

Second Embodiment

An embodiment of the present invention will be described below withreference to FIGS. 8 to 11.

FIG. 8 is a perspective view of a reaction chip in the presentembodiment. FIG. 9A is a plan view of the reaction chip and FIG. 9B is asectional view along a line A-A′ in FIG. 8. FIG. 10 is a processsectional view showing a reaction method using the reaction chipfollowing procedures thereof. FIG. 11 is a sectional view showinganother example of the reaction chip.

For convenience of description, it is assumed below that side of theresin base material positioned on the upper side, when a fluorescentreaction is detected or measured, is the “upper side” and the side ofmetallic base material positioned on the lower side is the “lower side”.

A reaction chip 21 in the present embodiment is a small chip that has,as shown in FIG. 8, a rectangular shape and a thickness of about severalmm. The reaction chip 21 is constituted by a cover material 22 (firstbase material) and a substrate 23 (second base material) embedded on theside of the undersurface of the cover material 22. The reaction chip 21in the present embodiment has, as shown in FIG. 9B, recesses 26constituting reaction containers 24 formed in the cover material 22 andgrooves 28 constituting recesses 27 and channels 25 formed in thesubstrate 23. The reaction chip 21 in the present embodiment includesthree sets of the channels 25 having the 12 reaction containers 24.

(Cover Material)

The cover material 22 presents a rectangular shape as a whole and isformed to such a thickness that the cover material 22 is not easily bentwhile being used. The cover material 22 is constituted by a resinmaterial such as PP (polypropylene), PC (polycarbonate), acryl resin(polymethyl methacrylate), PET (polyethylene terephthalate), PE(polyethylene), PV (polyvinyl chloride), and PS (polystyrene). The covermaterial 22 produced by using such a synthetic resin is preferable dueto superiority in heat resistance, chemical resistance, and moldingworkability. Further the cover material 22 produced by two types ofresin or more being bonded may be used. In such a case, various kinds ofthe cover materials 22 in accordance with properties of the reactionreagent or reagent solution can be produced by preparing the covermaterial 22 making the most of characteristics of each resin, so thatthe different cover materials 22 can be used for different purposes. Forexample, the material for the upper part and that for the lower part ofthe cover material 22 may be separated. Incidentally, in addition toresin materials, quartz glass or the like may be used as the material ofthe cover material 22.

The cover material 22 has, as shown in FIGS. 9A and 9B, a plurality (inthe present embodiment, 36) of recesses 26 in which a reagent isarranged and a reagent solution injecting hole 30 communicativelyconnected to the reaction container 24 and the channel 25 to feed areagent solution provided therein. In one set of the channel 25, aminute through hole 31 is provided at the end on the opposite side ofthe reagent solution injecting hole 30 and a high-density filter (notshown) is filled inside the through hole 31. Accordingly, a fed reagentsolution can be prevented from overflowing from an outlet.Alternatively, a similar reagent solution injecting hole may be providedat the end on the opposite side of the reagent solution injecting hole30 so that a reagent solution can be injected through whichever of thereagent solution injecting holes of the channel 25. The inner side ofthe reagent solution injecting hole 30 is preferably tapered so that thetip of a dispensing chip for general PIPETMAN fits in halfway throughthe injecting hole. Accordingly, feeding of a reagent solution is madeeasier and the mixing of bubbles can be prevented. Moreover,contamination of the apparatus due to scattering of a reagent solutionduring reaction can be prevented by providing a lid covering the reagentsolution injecting hole 30 and a structure on the outlet side.

(Substrate)

The substrate 23 presents a rectangular shape as a whole. The substrate23 is constituted by materials containing metal such as gold, silver,copper, aluminum, zinc, tin, platinum, nickel, brass, or alloys of atleast two of these metals. Producing the substrate 23 using materialscontaining such metals makes the thermal conductivity to a reactionliquid in the reaction container 24 higher so that the reaction canpreferably be caused to occur efficiently in a short time. Moreover, asealant layer (not shown) is provided in an upper part of the metalliclayer of the substrate 23 to stick the cover material 22 and thesubstrate 23 together by thermal welding. According to thisconfiguration, a metal that inhibits a reaction can also be used becausethe metal constituting the substrate 23 does not come directly intocontact with a reaction liquid.

The substrate 23 has a plurality (in the present embodiment, 36) ofrecesses 27 formed at positions corresponding to the recesses 26 of thecover material 22 and the grooves 28 to feed a reagent solution to allowcommunicative connection between the adjacent recesses 27. The diameterof the recess 27 is preferably almost the same as that of the recess 26of the cover material 22. Accordingly, a reaction reagent solution canbe fed equally to the recesses 26 and the recesses 27 and also themixing of bubbles can be prevented. The width and depth of the channel25 are preferably 0.5 mm or more and 5 mm or less. If the width anddepth are within these dimensions, channel blockage caused by extrusionof the sealant layer into the channel when the cover material 22 and thesubstrate 23 are stuck together can be prevented and also the mixing ofbubbles can be prevented.

(Reaction Container)

As shown in FIG. 10A, a lower area facing the substrate 23 of the recess26 on the cover material 22 side is a columnar space and an upper(bottom side) area is a truncated cone shaped space. As shown in thiscase, the bottom of the recess 26 is preferably flat. Accordingly, whena reaction result is obtained by fluorescence detection through thetransparent cover material 22, diffusion of light is reduced whencompared with a case where the bottom is not flat and fluorescence canefficiently be detected. The diameter of the recess 26 is preferably 0.5mm or more and 10 mm or less. Accordingly, feeding of a reagent solutionto the recess 26 is made easier and the mixing of bubbles can beprevented.

The recess 27 on the substrate 23 side has, as shown in FIG. 10A, ahemispheric shape. If the recess 27 in a lower part of the reactioncontainer 24 is formed in a hemispheric shape, convection is efficientlycaused inside the reaction container 24 when heat is added for areaction after a reagent solution being filled so that the reaction canbe made to proceed more smoothly. If the shape of the reaction container24 is formed into a shape similar to that of a tube made of PP generallyused for PCR, adhesion property to a heat block that adds heat for areaction is increased, so that heat can be transmitted efficiently to areaction liquid, allowing the reaction to proceed in a short time.

The recess 26 is formed by a method of cutting the cover material 22made of resin material or a method of injection-molding a resin materialinside a die. If the cover material 22 is constituted by a hard resinmaterial such as PC (polycarbonate), the recess 26 can be formed usingthe cutting method. If the cover material 22 is constituted by a softresin material such as PP (polypropylene), the recess 26 is preferablyformed using the molding method. The recess 26 can also be formed fromPC using the molding method.

On the other hand, the recess 27 and the groove 28 are formed by amethod of performing drawing using a die on the substrate 23 in which ametallic layer and a sealant layer are stuck together by an adhesive orthe like.

(Sealing Compound)

As shown in FIG. 10A, a fixing reagent S such as a nucleic acid probe isarranged inside the recess 26 of the cover material 22. The fixingreagent S is covered with a hot-melt sealing compound W arranged insidethe recess 26. The hot-melt sealing compound W is a sealing compoundthat is in a solid state at ordinary temperature and melts near astarting temperature of a reaction of the fixing reagent and a reagentsolution (hereinafter, referred to as a “main reaction”). The meltingpoint thereof is preferably near 35 to 90° so that sealing compound Wmelts at least near 80 to 90°. It is preferable to adopt a sealingcompound whose specific gravity is smaller than that of the fixingreagent and also that of the reagent solution as the sealing compound W.However, the premise is that the main reaction is not inhibited. As aconcrete sealing compound, AmpliWax (registered trademark) PCR Gem 100manufactured by Applied Biosystems can be adopted. This is a productinvented as a replacement of mineral oil so that evaporation of areaction reagent solution is prevented by forming a layer after meltingwhen a PCR amplification reaction occurs. This product is in a solidstate at ordinary temperature and melts at 55 to 58°. The fixing reagentS covered with the sealing compound W may be in a liquid state or asolid state. If the fixing reagent S is in a liquid state whose specificgravity is larger than that of the reagent solution, or the meltedsealing compound W has a specific gravity larger than that of the fixingreagent S and that of the reagent solution, it is easier and moreadvantageous to mix the fixing reagent S and the reagent solution.

To arrange the sealing compound W inside the recess 26, a method ofinjecting a proper amount of the solid sealing compound W into therecess 26 in which the fixing reagent S is pre-arranged and heating thesealing compound W can be used.

Accordingly, the melted sealing compound W spreads at the bottom of therecess 26 while wetting the bottom and, if the sealing compound W iscooled thereafter, the sealing compound W can be arranged inside therecess 26 while the fixing reagent S being covered therewith.Alternatively, a method of dispensing the pre-melted sealing compound Winto the recess 26 in which the fixing reagent S is pre-arranged usingPIPETMAN may be used. This method is more advantageous because theamount of the sealing compound W can be defined more accurately.

Further, it is preferable to perform a centrifugal operation beforecooling the melted sealing compound W while wetting and spreading at thebottom of the recess 26. Accordingly, the sealing compound W can hidethe fixing reagent S more reliably. Moreover, the sealing compound W onthe wall surface of the recess 26 moves to the bottom of the recess 26and thus, when the cover material 22 and the substrate 23 are stucktogether by thermal welding, the outflow of the sealing compound W tothe channel 25 due to re-melting can be prevented.

In this manner, the sealing compound W is arranged inside the recess 26before the substrate 23 being stuck together.

(Reaction Method)

Next, the reaction method using the above reaction chip will bedescribed using FIGS. 8 to 10.

First, the reagent solution L is injected through the reagent solutioninjecting hole 30 shown in FIGS. 8 and 9. In this manner, as shown inFIG. 10B, the reagent solution L is passed from the reagent solutioninjecting hole 30 into the channel 25. Then, the reagent solution Lpasses through the channel 25 before being fed into a plurality of thereaction containers 24 one by one. The feeding of the reagent solution Loccurs at ordinary temperature or a lower temperature below the ordinarytemperature at which the reagent solution L can be fed.

Here, as shown in FIG. 10B, the solid sealing compound W in a statecovering the fixing reagent S is arranged inside the recess 26 on thecover material 22 side. Thus, the reagent solution L fed to the reactioncontainer 24 is arranged on the surface of the sealing compound Wwithout coming into contact with the fixing reagent S.

Thus, in the reaction chip 21 in the present embodiment, the reagentsolution L is fed below the sealing compound W covering the fixingreagent S, so that the fixing reagent S will not flow out to theadjacent reaction containers 24. Therefore, contamination can beprevented from occurring.

After the reagent solution L being fed into the reaction container 24,the channel 25 is blocked by plastically deforming a part of the groove28 between the adjacent recesses 27 of the substrate 23 to isolate eachof the reaction containers 24. By isolating each of the reactioncontainers 24, mixing of unnecessary reagents between the adjacentreaction containers 24 can be prevented.

As means for plastic deformation of the groove 28 of the substrate 23,an external force may mechanically be applied to apart of the groove 28from outside by using a device or an external force may be applied byhand.

Next, as shown in FIG. 10C, the reaction container 24 is heated to meltthe sealing compound W. At this point, heat is added not from the covermaterial 22 side where the fixing reagent S is arranged, but from thesubstrate 23 side. If the specific gravity of the sealing compound W issmaller than that of the fixing reagent S and also that of the reagentsolution L, the sealing compound W changes places with the fixingreagent S vertically when melted and the fixing reagent S comes intocontact with the reagent solution L. If the specific gravity of thesealing compound W is larger than that of the fixing reagent S and alsothat of the reagent solution L, the sealing compound W changes placeswith the reagent solution L vertically when melted and the reagentsolution L comes into contact with the fixing reagent S. If the reactioninitiation temperature of the main reaction is equal to the meltingpoint of the sealing compound W or higher than the melting point of thesealing compound W, the fixing reagent S and the reagent solution L comeinto contact while being heated to the reaction initiation temperatureand the main reaction is initiated when the reaction initiationtemperature is reached.

If the reaction initiation temperature of the main reaction is lowerthan the melting point of the sealing compound W, the reaction container24 is further heated to bring the fixing reagent S into contact with thereagent solution L and then, the temperature is lowered to the reactioninitiation temperature to initiate the main reaction. In the presentembodiment, heat is added during reaction from the substrate 23 side.

According to the reaction method in the present embodiment, the reactionchip 21 is constituted by the cover material 22 made of resin having arelatively low thermal conductivity and the substrate 23 made of metalhaving a relatively high thermal conductivity and the fixing reagent Sis arranged inside the recess 26 of the cover material 22. When areaction is caused, heat is added from the substrate 23 side with a highthermal conductivity and the sealing compound W is melted to bring thefixing reagent S and the reagent solution L into contact before causingthe reaction to proceed. Therefore, when the reagent solution L is fed,the reagent S is covered with the sealing compound W so thatcontamination can be prevented from occurring. While thermal efficiencyfor the whole reaction container 24 can be made better by adding heatfrom the substrate 23 side, the reagent S is arranged on the covermaterial 22 side with a low thermal conductivity and thus, it is hardfor heat during chip manufacturing to conduct to the reagent S so thatactivity of the reagent S is neither lowered nor devitalized.Accordingly, accurate reaction data can be measured.

The technical scope of the present invention is not limited to the aboveembodiment and various modifications can be made without deviating fromthe spirit of the present invention. For example, the recess 27constituting the reaction container 24 and the groove 28 constitutingthe channel 25 are formed on the substrate 23 side in the aboveembodiment, but as shown in FIG. 11, a groove 33 may be formed on acover material 22A side so that a flat plate is used as a substrate 23Ain accordance with the volume necessary for the reaction container.Alternatively, the grooves 28 constituting the channel 25 are formedonly in the substrate 23 in the above embodiment, grooves may also beformed on the cover material 22 side in accordance with the volume ofthe reagent solution L so that the channel may be constituted by boththe cover material 22 and the substrate 23. Moreover, concreteconfigurations such as the shape, number, and arrangement of thereaction containers and channels, materials and dimensions of each basematerial, various methods used in each manufacturing process exemplifiedin the above embodiment are only examples and may be changed whenappropriate.

Third Embodiment

A temperature controlling unit for a gene treating apparatus(hereinafter, referred to as a “temperature controlling unit”) accordingto an embodiment of the present invention will be described below withreference to FIGS. 12 to 19.

FIG. 12 is a perspective view showing principal parts of a geneamplifying apparatus (gene treating apparatus) 42 including atemperature controlling unit 41 in the present embodiment. The geneamplifying apparatus 42 includes a movable carriage 43 on which areaction container is placed, the temperature controlling unit 41 thatheats or cools the reaction container, and a measuring unit 44 thatmeasures a reaction in the reaction container.

The movable carriage 43 is formed in a frame shape and the reactioncontainer described later is mounted thereon with the undersurfacethereof exposed. The movable carriage 43 is configured to be able tomove above the temperature controlling unit 41 along a rail 46 set up onthe top face of the gene amplifying apparatus 42 by a moving unit 45composed of a publicly known configuration such as a stepping motor anda servo motor.

In addition to the above configuration, the moving unit 45 can be usedby appropriately selecting from configurations of publicly known movingunits, for example, a combination of a stepping motor and a belt or aconfiguration in which the rail 46 and the movable carriage 43 are movedin a non-contact fashion by using a magnetic force or the like.

The measuring unit 44 is constituted by an emission detection unit 47that introduces excitation light and measures fluorescence and ameasuring unit moving unit 48 that moves the emission detection unit 47,and carries out an inspection of a gene sample after being amplified.The measuring unit 44 is not indispensable for the gene amplifyingapparatus 42 in the present invention and may not be provided if theconfiguration is intended for amplification only.

The temperature controlling unit 41 is constituted by a first unit 49arranged above the movable carriage 43 and a second unit 50 arrangedbelow the movable carriage 43. The first unit 49 is vertically movablysupported by a pair of support arms 51. The second unit 50 is alsovertically movably supported by a moving unit (not shown).

With the above configuration, the temperature controlling unit 41 isconfigured so that the first unit 49 and the second unit 50 moves towardthe movable carriage 43 stopped between the first unit 49 and the secondunit 50 by moving on the rail 46 to be able to sandwich the movablecarriage 43 and the reaction container placed on the movable carriage 43for heating/cooling.

FIG. 13 is a schematic sectional view showing a state where a reactioncontainer 100 is sandwiched by the temperature controlling unit 41 andFIG. 14 is an enlarged sectional view showing details near the reactioncontainer 100 in FIG. 13. In FIGS. 13 and 14, units such as the movablecarriage 43 and the rail 46 are omitted to make the configuration of thetemperature controlling unit 41 easier to understand.

As shown in FIG. 13, the first unit 49 includes a first temperaturecontrolling unit 52 that heats/cools the top face side of the reactioncontainer 100 and a first heat sink (first heat dissipation unit) 53provided above the first temperature controlling unit 52 in contact withthe first temperature controlling unit 52. Similarly, the second unit 50includes a second temperature controlling unit 54 that heats/cools theundersurface of the reaction container 100 and a second heat sink(second heat dissipation unit) 55, and the first unit 49 and the secondunit 50 have the temperature controlling units 52, 54 arranged oppositeto each other respectively.

Each of the temperature controlling units 52, 54 is composed of aPeltier module and heats/cools the reaction container 100 by beingenergized by a power supply (not shown). As shown in FIG. 14, each ofthe temperature controlling units 52, 54 has a first heat conductionlayer 56 made of carbon graphite to improve thermal conductivityprovided on upper and lower sides thereof.

Each of the heat sinks 53, 55 is a publicly known air-cooled heat sinkclosely provided with a fan 57 and dissipates heat generated by each ofthe temperature controlling units 52, 54 out of the apparatus. Insteadof an air-cooled heat sink, a water-cooled heat sink may be provided.

Each of the units 49, 50 is connected to a control unit 58 that sets andcontrols the temperature each of the temperature controlling units 52,54. The control unit 58 may be embedded in the gene amplifying apparatus42 or accommodated in a device such as an external personal computerconnected to the gene amplifying apparatus 42. The mode of temperaturecontrol of the control unit 58 will be described later.

As shown in FIG. 14, a pair of metallic plates 59 to uniformly dissipateheat generated by each of the temperature controlling units 52, 54 in asurface direction are arranged on the first heat conduction layer 56 onthe side of each of the temperature controlling units 52, 54 in contactwith the reaction container 100. Silver, aluminum or the like can beadopted as the material of the metallic plate 59.

The above first heat conduction layer 56 is provided on the side of themetallic plate 59 facing the reaction container 100 and a pair of secondheat conduction layers (heat conduction members) 60 made of thermalconductive material having elasticity. A silicon rubber sheet (tradename: Sarcon, manufactured by Fuji Polymer) having a high thermalconductivity, a silicone gel sheet (trade name: λGEL, manufactured byGeltec) having a high thermal conductivity or the like can be adopted asthe sheet material constituting the second heat conduction layer 60.

The second heat conduction layer 60 mounted on the second unit 50 ispreferably made thicker slightly so that the second heat conductionlayer 60 is able to be in contact with the entire surface of thereaction container 100 even if the lower part of the reaction container100 is uneven. In the present embodiment, the thickness of the secondheat conduction layer 60 mounted on the first unit 49 to 0.5 mm and thatof the second unit 50 to 2.0 mm. If the upper part of the reactioncontainer 100 is uneven, countermeasures can be taken by making thesecond heat conduction layer 60 mounted on the first unit 49 thicker.

With the above configuration, each of the temperature controlling units52, 54 heats and cools the entire top face and undersurface of thereaction container 100 via the first heat conduction layer 56, themetallic plates 59, and the second heat conduction layer 60.

Outer circumferences of each of the temperature controlling units 52, 54and the reaction container 100 are covered with a heat insulatingmaterial 61. Resin, Styrofoam or the like can be adopted as the heatinsulating material 61.

FIG. 15 is a perspective view exemplifying the reaction container 100used in the gene amplifying apparatus 42, FIG. 16 is a sectional viewalong a line A-A′ in FIG. 15, and FIG. 17 is a sectional view along aline B-B′ in FIG. 15.

As shown in FIGS. 15 and 16, the reaction container 100 is constitutedby a first member 101 made of resin and arranged in the upper part and asecond member 102 made of metal and arranged in the lower part.

Polypropylene or the like can be adopted as the first member 101 andaluminum, copper or the like can be adopted as the second member 102.

As shown in FIG. 15, the reaction container 100 has a plurality of wells103 filled with a gene sample containing genes (nucleic acid). That is,the upper part of each of the wells 103 is formed of the first member101 and the lower part thereof is formed of the second member 102 andthus, thermal conductivity is different in the upper part and the lowerpart of each of the wells 103, with the upper part having a lowerthermal conductivity.

A reagent 104 used for PCR reaction is arranged on the inner surface ofthe first member constituting the upper part of the well 103.Incidentally, instead of the reagent 104 being arranged inside the well,the well may be filled with the reagent 104 together with a gene sampledescribed later.

As shown in FIGS. 15 and 17, the wells 103 are communicatively connectedby a channel 105 in groups of any number and a injecting hole 106 toinject a gene sample and a deaeration port 107 are provided at both endsof each of the channels 105. When a gene sample is injected through theinjecting hole 106, the air inside the channel 105 is exhausted throughthe deaeration port 107, and the gene sample passes through the channel105 before each of the communicatively connected wells 103 being filledtherewith.

The operation when the gene amplifying apparatus 42 configured asdescribed above is used will be described below.

First, the channel 105 of the reaction container 100 filled with a genesample is by a jig or the like to make each of the wells 103 anindependent space. Then, the reaction container 100 is placed on themovable carriage 43 and the gene amplifying apparatus 42 is started.

The reaction container 100 on the movable carriage 43 is moved on therail 46 by the moving unit 45 before being stopped between the firstunit 49 and the second unit 50 of the temperature controlling unit 41.After the movable carriage 43 being stopped, the first unit 49 falls andthe second unit 50 rises before the reaction container 100 beingsandwiched from above and from below by the temperature controlling unit41 so that the first unit 49 and the second unit 50 comes into contactwith the entire top face and undersurface of the reaction container 100respectively.

After the reaction container 100 being sandwiched by the temperaturecontrolling unit 41, the first temperature controlling unit 52 and thesecond temperature controlling unit 54 are energized by a power supply(not shown). Then, the reaction container 100 is heated and cooled toreach a predetermined temperature cycle under the control of the controlunit 58 to amplify a gene contained in the gene sample inside thereaction container 100 by the PCR method.

FIG. 18 is a graph showing a temperature cycle by the PCR method,temperature control by the temperature controlling unit 41, andtemperatures of each unit of the reaction container. The control unit 58exercises independent temperature control for the first temperaturecontrolling unit 52 and the second temperature controlling unit 54.

In the present embodiment, as shown in FIG. 18, gene amplification bythe PCR method is carried out, as indicated by a thick solid line, in atemperature cycle in which temperatures near 95° and 68° are mutuallyrepeated. Therefore, this temperature cycle becomes a target temperaturefor the gene sample and the reference for temperature control by each ofthe temperature controlling units 52, 54.

The lower part of the reaction container 100 is formed of the secondmember 102 having a high thermal conductivity and thus, by heating thesecond temperature controlling unit 54, as indicated by an alternatelong and short dash line, up to about 95° C., the temperature inside thewell 103 (near the central part in the vertical direction) also rises,as indicated by a broken line, up to close to 95° C. However, the upperpart of the reaction container 100 is formed of the first member 101whose thermal conductivity is lower than that of the second member 102and therefore, the temperature of the gene sample near the first member101 may not rise up to close to 95° C. necessary for PCR reaction. Insuch a case, the PCR reaction may not proceed or proceeds onlyinsufficiently.

Thus, as indicated by a chain double-dashed line, the preset temperatureof the first temperature controlling unit 52 is set to about 105° C.,which is higher than the target temperature 95° C. Accordingly, asindicated by a thin solid line, the surface temperature of the firstmember 101 rises to 95° C. or higher so that it is supposed that thetemperature inside the well 103 becomes uniform as a whole and also thefact that temperature changes proceed quickly along the presettemperature cycle was confirmed.

Actually, with the above temperature settings, the PCR reaction insidethe reaction container 100 proceeded satisfactorily so that 35 cyclescould be completed in about 41 minutes, which is about half the timethat was needed with a conventional apparatus.

FIG. 19 is a graph showing, as an example of a conventional apparatus,temperature changes when the reaction container 100 is used in a geneamplifying apparatus that heats/cools the reaction container 100 by aPeltier module being brought into contact with only the lower partthereof. While the temperature inside the well rises up to close to 95°C. by the Peltier module, the surface temperature of the first member101 rises only to close to 80° C. so that it is supposed that thetemperature inside the well is non-uniform. Actually, cases where thePCR reaction did not proceed in this gene amplifying apparatus wereconfirmed.

In this apparatus, it seems that quite a long time will be needed tobring the surface temperature of the first member 101 closer to thetarget temperature as far as a temperature profile is concerned and itis assumed that the total time necessary for PCR reaction will be verylong.

On the other hand, even if an attempt is made to reduce the PCR time byrapid heating and rapid cooling by setting the control temperature ofthe Peltier module higher than the target temperature when thetemperature rises and lower when the temperature falls, a region wherethe temperature inside the well (particularly near the lower part)follows behavior of the control temperature arises because theundersurface of the reaction container 100 is composed of the secondmember 102 having a high thermal conductivity so that a case where thereagent inside the well is devitalized can be considered.

Actually, a case where the PCR reaction did not proceed when suchcontrol was exercised is confirmed and devitalization of the reagent wassuggested as one of possible causes why the PCR reaction did notproceed.

The temperature control of the temperature controlling unit 41 by thecontrol unit 58 described above is only an example and settingparameters such as the actual preset temperature and lengths of theheating/cooling time (the time during which the preset temperature ismaintained) are independently decided for the first temperaturecontrolling unit 52 and the second temperature controlling unit 54depending on reaction container parameters such as the thermalconductivity, thickness and the like for each material of the firstmember 101 and the second member 102.

Thus, not only the preset temperature, but also the heating/cooling timemay be different for the first temperature controlling unit 52 and thesecond temperature controlling unit 54. Therefore, setting parametersfor each reaction container parameter may be stored in the control unit58 as a table in advance so that, based on user input or the like, thecontrol unit 58 exercises temperature control of the first temperaturecontrolling unit 52 and the second temperature controlling unit 54 byreferring to corresponding setting parameters in the table whenappropriate.

The reaction container 100 whose amplification is completed moves up tothe measuring unit 44 along the rail 46. Then, various measurements suchas fluorescence intensity of the sample in each of the wells 103 aremade by the measuring unit 44. A sample amplified by the gene amplifyingapparatus 42 of the present invention can be offered to various genetictests such as a base sequence test of a gene, polymorphism test based ona repetitive sequence with a certain base sequence set as a unit, andtest of single nucleotide polymorphism (SNPs or SNP).

According to the temperature controlling unit 41 and the gene amplifyingapparatus 42 in the present embodiment, heat generated by the firsttemperature controlling unit 52 and the second temperature controllingunit 54 is efficiently transmitted to the reaction container 100 by thefirst heat conduction layer 56, the metallic plates 59, and the secondheat conduction layer 60. Thus, even if the reaction container 100 isformed by including the first member 101 and the second member 102having different thermal conductivities, a filled gene sample isappropriately heated/cooled and a time required to realize a temperaturecycle necessary for the PCR method is reduced so that the gene can beamplified quickly and suitably.

Moreover, the control unit 58 exercises temperature control of the firsttemperature controlling unit 52 and the second temperature controllingunit 54 independently in accordance with various parameters includingthermal conductivities of the members 101, 102 of the reaction container100 so that each of the first temperature controlling unit 52 and thesecond temperature controlling unit 54 is controlled to the optimumpreset temperatures and heating/cooling times to set the gene sample attemperatures in keeping with the temperature cycle of PCR. Therefore,the gene sample filled inside the reaction container 100 can bePCR-treated more suitably.

Moreover, the circumference of each of the temperature controlling units52, 54 sandwiching the reaction container 100 is covered with the heatinsulating material 61 and thus, heat exchanged between each of thetemperature controlling units 52, 54 and the reaction container 100 doesnot escape to the outside so that the temperature of the reactioncontainer 100 can be controlled more efficiently.

Further, the first heat sink 53 and the second heat sink 55 are providedin contact with the first temperature controlling unit 52 and the secondtemperature controlling unit 54 respectively and thus, when the reactioncontainer 100 is cooled, heat transmitted to each of the temperaturecontrolling units 52, 54 can efficiently be dissipated out of thetemperature controlling unit 41.

In the foregoing, an embodiment of the present invention has beendescribed, but the technical scope of the present invention is notlimited to the above embodiment and various modifications can be madewithout deviating from the spirit of the present invention.

For example, an example in which the first heat conduction layer 56 isprovided in contact with each of the temperature controlling units 52,54 and the metallic plates 59 is described in the above embodiment, butthe arrangement position of the first heat conduction layer 56 is notlimited to this. Other examples may include providing the first heatconduction layer 56 in contact with only each of the temperaturecontrolling units 52, 54 and in contact with only the metallic plates59.

Moreover, if the temperature is controlled satisfactorily, the firstheat conduction layer 56 need not necessarily be provided.

The reaction container to be used is not limited to, as described above,a reaction container in which the thermal conductivity of the upper partis lower than that of the lower part and, for example, a reactioncontainer in which the thermal conductivity of the upper part is higherthan that of the lower part may also be adopted. In such a case, the PCRreaction can be caused to proceed more suitably by changing the controlmode of the control unit.

In a temperature controlling unit and a gene treating apparatus of thepresent invention, the control unit is not required. For example, if thedifference in thermal conductivity between first and second members isrelatively small and the PCR reaction can be caused to proceed withoutindependent temperature control of first and second temperaturecontrolling units, the temperature controlling unit and the genetreating apparatus may be configured without providing a control unit.

Further, units such as a movable carriage and a rail to move a reactioncontainer are not required in a gene treating apparatus of the presentinvention. For example, the gene treating apparatus may be configured insuch a way a reaction container is directly set up between the firstunit 49 and the second unit 50 by the user and the temperature iscontrolled for PCR reaction at the setup position.

In addition to an amplification reaction by the PCR method, atemperature controlling unit and a gene treating apparatus of thepresent invention can also be used when a predetermined temperature ismaintained for a fixed time like, for example, a reaction by the invadermethod. In such a case, the temperature of a gene sample in a reactioncontainer can suitably be controlled without being affected by adifference in thermal conductivity of members constituting the reactioncontainer.

According to a reaction chip of the present invention, a notch showing agradual increase in width and a gradual increase in depth from one faceof the base material toward an inner wall surface of the recess isformed on an edge of at least one recess of the recesses in an extendingdirection of the groove and therefore, the flow of a reagent solutionbecomes smooth near the recess so that the reagent solution smoothlyflows into the recess constituting a reaction container from a grooveconstituting a channel or flows out to the groove from the recess. Thus,even a reagent solution containing bubbles comes flowing, the frequencywith which bubbles remain inside the recess by being entrapped by theinner wall surface of the recess can significantly be decreased.Therefore, the desired reaction can accurately be detected and measuredby using a reaction chip of the present invention. Moreover, bubbles canbe removed from inside the recess simply by molding a notch on an edgeof the recess and therefore, there is no need of hydrophobic/hydrophilictreatment and surface treatment such as corona treatment and plasmatreatment.

If the configuration is adopted in which the angle formed by one face ofthe base material and the notch is smaller than that formed by one faceof the base material and the inner wall surface of the recess, theinclination of the inner wall surface on the inflow side or outflow sideof the recess becomes gentle, which makes the flow of the reagentsolution in a sectional direction of the base material smooth, so thatbubbles can effectively be prevented from remaining.

If a notch is formed only on one side of the extending direction of thegroove and the configuration is adopted in which the notch is formed onthe inflow side of the reagent solution, the reagent solution flows intothe recess from the groove smoothly so that bubbles can effectively beprevented from remaining.

If a notch is formed also on the outflow side of the reagent solutionand the configuration in which the notch formed on the inflow side ofthe reagent solution and the notch formed on the outflow side of thereagent solution form a line symmetric shape is adopted, two notches canbe formed easily and also the flow of the reagent solution becomessmooth, so that bubbles can more effectively be prevented fromremaining.

If the recess has a columnar space having the inner wall surface atsubstantially right angles to one face of the base material on anopening side and the maximum depth on the edge of the notch is shallowerthan the depth of the columnar space, the inner wall surface atsubstantially right angles in the columnar space will remain on the edgeof the recess on the side on which the notch is formed. By using thisinner wall surface, a reagent or a fixing agent that temporarily fixesthe reagent can reliably be accommodated, so that reaction products canbe prevented from leaking to adjacent reaction containers.

If an outer shape of the recess is circular in plane view and theconfiguration is adopted in which a plane shape of the notch is defined,when two tangents to a circle forming an outer edge of the recess aredrawn from one point on one face of the base material in the extendingdirection of the groove, by an area inside the two tangents, an overallshape of the recess including the notch has a shape with the least flowresistance and the flow of the reagent solution near the recess becomesextremely smooth, so that bubbles can more reliably be prevented fromremaining.

If a center line in the extending direction of the groove of the notchis aligned with the center line of the groove on the same straight line,the flow of the reagent solution becomes smooth without being deviatedinside the recess, so that bubbles can more reliably be prevented fromremaining.

According to a reaction method of the present invention, a reaction chipis constituted by a first base material with a relatively low thermalconductivity, and a second base material with a relatively high thermalconductivity and a reagent is arranged inside a recess of the first basematerial. Then, when a reaction is caused, heat is added from the sideof the second base material with a higher thermal conductivity and thereagent and a reagent solution are brought into contact by melting asealing compound to cause the reaction to proceed. Therefore, when thereagent solution is fed, the reagent is covered with the sealingcompound so that contamination can be prevented from occurring. Whilethermal efficiency for the whole reaction container is excellent byadding heat from the side of the second base material, the reagent isarranged on the side of the first base material with a lower thermalconductivity and therefore, it is hard for heat added during chipmanufacturing to be transmitted to the reagent so that activity of thereagent will be neither lowered nor devitalized. Accordingly, accuratereaction data can be measured.

If the configuration in which heat is added from the side of the secondbase material and the first base material and the second base materialare stuck together by thermal welding of a sealant layer provided on atleast one of the first base material and the second base material isadopted, it is hard for the sealing compound to melt by heat when thebase materials are stuck together, malfunctions such as blockage of thechannel due to outflow of the sealing compound and incomplete sealing ofthe reagent can be prevented so that a reaction chip can be producedwith stability and also contamination can reliably be prevented fromoccurring. If a sealant layer that does not inhibit a reaction is used,the material of each base material can freely be selected. Accordingly,the material capable of realizing a chip having high heat resistance,barrier property, chemical resistance, and reagent preservability andsuperior in reactivity (thermal conductivity) can be selected.

If a recess corresponding to the recess of the first base material isformed also on the second base material to configure the reactioncontainer by both the recess of the first base material and that of thesecond base material, a sufficient volume of the reaction container canbe ensured and also flexibility of design for the volume and shape ofthe reaction container can be increased. Moreover, the surface area ofthe second base material with a higher thermal conductivity increases,which increases the thermal conductivity for the whole reactioncontainer, so that a reaction that proceeds by heating such as an enzymereaction can be caused more efficiently in a short time.

If the configuration in which a resin material is used as the first basematerial and a metallic material is used as the second base material isadopted, a reaction container or channel having the above excellentcharacteristics can easily be worked on.

If the sealing compound is constituted by a material soluble in neithera reagent nor a reagent solution, the reagent and the reagent solutioncan come into contact and react without any change in compositionthereof so that accurate reaction data can be measured.

If the reaction container is a reaction container for enzyme reaction, aDNA amplification reaction by an enzyme reaction, DNA detection reactionby hybridization, and detection reaction of SNP, which are generalbiochemical reactions, can be realized on a reaction chip.

According to a temperature controlling unit for gene treating apparatusand a gene treating apparatus of the present invention, even if areaction container constituted by plural types of materials havingdifferent thermal conductivities, a gene contained in a gene samplefilled in the reaction container can suitably be treated.

EXAMPLES

The present inventors carried out experiments below to demonstrateeffects of the first embodiment of the present invention.

Example 1

First, the resin base material 2 including the recess 6 having the shapeand layout shown in FIGS. 2 and 4 according to the above embodiments isproduced by injection molding. Polypropylene determined not to inhibit areaction is used as a material. As for the notch 15, after the resinbase material 2 including the recess 6 being produced by using injectionmolding, the notch 15 is formed by cutting at upstream and downstreampositions of the channel 5 on the edge of the recess 6. On the otherhand, what is produced by performing drawing on an aluminum originalsheet to which a polypropylene sealant is applied is used as themetallic base material 3 with the intention of improving thermalefficiency during reaction.

While the reaction chip has a reagent arranged in each of the reactioncontainers 4 in a normal case, the present experiment is intended tocheck the state of bubbles during feeding and thus, instead of areagent, 5 μl of AmpliWax (trade name, manufactured by ABI) is each putinto the recess 6 of the resin base material 2. The resin base material2 having AmpliWax arranged inside the recess 6 and the metallic basematerial 3 are welded by thermal welding to produce a reaction chip inthe present embodiment.

Comparative Example 1

A resin base material 2A including the recess 6 having no notch as shownin FIGS. 20A and 20B is produced and then, a reaction chip ofComparative Example 1 is produced by following the same method as thatof Example 1 described above (see FIGS. 21A and 21B).

As reagent solutions to be fed, a reagent solution A obtained bydiluting a PCR product 10 times and a reagent solution B composed of a10 mg/ml protein solution (BSA solution) are prepared. These reagentsolutions A and B are fed to three reaction chips each for Example 1 andComparative Example 1 by an electric pipette (Finnpipette Novas 30-300μl (trade name), manufactured by Thermo Fisher Scientific) at the flowrate of 200 μl/21 sec.

If a reagent solution is fed, bubbles contained in the reagent solutionare introduced into each reaction container, but in reaction chips ofExample 1, bubbles once introduced flow out together with the reagentsolution and no bubble remains inside the reaction container. FIG. 22shows a photo shooting conditions inside the reaction container througha transparent resin base material.

In reaction chips of Comparative Example 1, on the other hand, bubblesonce introduced do not flow out of the reaction container and, as shownin FIG. 21C, a bubble B remaining inside the reaction container 4 isobserved. FIG. 23 shows a photo shooting conditions inside the reactioncontainer.

Numbers of reaction containers in which bubbles remain during feeding ineach of reaction chips of Example 1 and Comparative Example 1 are listedin [Table 1]. The total number of reaction containers is 108 becausethree reaction chips each with 36 reaction containers are fed. While thenumber of reaction containers in which bubbles remain is 0 for both thereagent solution A and the reagent solution B in reaction chips ofExample 1, the number of reaction containers in which bubbles remain is8 for the reagent solution A and 18 for the reagent solution B inreaction chips of Comparative Example 1.

TABLE 1 Reagent solution Reagent solution Reaction A PCR diluted BProtein container shape solution solution Example 1 With notch 0 0Comparative Without notch 8 18 Example 1

From the above result, an effect of the reaction chip of the presentinvention having a recess with notch is proved.

The detection method of SNP using the invader (registered trademark)method executed by the present inventors as Example 2 of a reaction chipand a reaction method according to the second embodiment of the presentinvention will be described below. FIG. 24A is a plan view showingreagent arrangement of Example 2 and Comparative Example 2, FIG. 24B isa sectional view of the reaction chip of Example 2, and FIG. 24C is asectional view of the reaction chip of and Comparative Example 2.

Example 2

As shown in FIG. 24A, a reaction chip described in the above embodimentis produced. The cover material 22 is produced by the method ofinjection-molding polypropylene resin inside a die and a plurality ofrecesses 26 and a plurality of reagent solution injecting holes 30 areformed on a resin base material with a thickness of 2 mm. The openingdiameter of the recess 26 is 3 mm, the diameter thereof at the bottom is2 mm, and the depth thereof is 1.5 mm. The volume of the recess 26 istheoretically about 9 μL and the distance between the adjacent recesses26 is 6 mm. The outside diameter of the reagent solution injecting hole30 is 4 mm and the inside diameter of the hole is 1.5 mm to 2 mm,creating a tapered shape. The height thereof is 6 mm from the top faceof the cover material 22.

A material in which a sealant layer made of polypropylene of 70 μm isstacked on an aluminum plate of 0.1 mm via an adhesive is used as thesubstrate 23 and the channel 25 communicatively connecting the recess 27and the recess 27 is formed by drawing. The opening diameter of therecess 27 is 3 mm and the depth thereof is 1.5 mm. The volume of therecess 27 is theoretically about 7 μL and the distance between theadjacent recesses 27 is 6 mm. The width of the channel 25 is 1 mm andthe depth thereof is 0.3 mm.

As shown in FIG. 24B, the fixing reagent S is arranged in the recess 26of the cover material 22 corresponding to shaded reaction containers 4Sin FIG. 24A. An allele probe 1, an allele probe 2, and an invader probeused for an invader (registered trademark) reaction and an FRET probe 1,an FRET probe 2, and Cleavase (registered trademark) are arranged as thefixing reagents S before heating/drying.

Next, AmpliWax (registered trademark) PCR Gem 100 manufactured byApplied Biosystems is put into all the recesses 26 as the sealingcompound W. The amount of the sealing compound W for one recess 26 is4.5 μL. The sealing compound W is heated to 80 to 100° C. to bedispensed to the recess 26 while being melted and, after a centrifugaloperation being performed by a plate centrifuge, the sealing compound Wis solidified again at ordinary temperature. Accordingly, the fixingreagent S is covered with the sealing compound W in the shaded reactioncontainers 4S in FIG. 24A.

Next, the cover material 22 and the substrate 23 produced above arestuck together by a heat seal under the conditions of 250° C., 0.5 MPa,1.4 s, and one-sided heating from the substrate 23 side using a heatseal tester manufactured by Tester Sangyo.

Next, a mixed solution of a PCR product amplified from purified genomeDNA, an invader buffer, and water for dilution thereof is fed as areaction reagent solution. The reaction reagent solution L is caused toreach all the reaction containers 24 through the channel 25 from thereagent solution injecting hole 30 of the cover material 22. The amountof feeding of the reaction reagent solution L to one reaction container24 is 12 to 15 μL.

Then, the reaction chip 21 is set to a developed analysis chip dedicatedapparatus. In the apparatus, a part of the channel 25 is crushed by anexternal force for sealing between the reaction containers 24 and 4S sothat a reaction liquid during reaction should not be exchanged betweenthe reaction containers 24. At this point, heat is added simultaneouslywith the external force for thermal welding of the sealant layer of thecover material 22 and the substrate 23 to strengthen sealing. Thesealing occurs under the conditions of 190° C., 120 kgf, and 1.5 s.

Next, the reaction chip 21 is heated from the substrate 23 side insidethe apparatus to cause a reaction. First, while the sealing compound Wis melted under the conditions of 95° C. and 5 minutes to bring thefixing reagent S and the reaction reagent solution L into contact, thePCR product in the reaction reagent solution is altered in quality.Then, the temperature is lowered to 63° C. and a fluorescent substanceis detected inside the reaction containers 4S are detected once in every30 seconds while causing an invader reaction to observe reactionconditions at regular intervals.

Comparative Example 2

The cover material 22 and the substrate 23 are produced in the samemanner as the above Example 2. Then, in Comparative Example 2, as shownin FIG. 24C, the fixing reagent S and the sealing compound W arearranged in the recess 27 of the substrate 23. After the cover material22 and the substrate 23 being stuck together by a heat seal, thereaction reagent solution L is input through the reagent solutioninjecting hole 30 at ordinary temperature. Next, after being sealedbetween the reaction containers 24 and 4S in the apparatus, the reactionchip 21 is heated from the substrate 23 side to melt the sealingcompound and bring the fixing reagent and a reaction reagent solutioninto contact and then, a fluorescent substance is detected while aninvader reaction being caused.

(Experiment Results)

FIGS. 25A and 25B show results of reactions caused in Example 2, andFIG. 25A shows luminescence intensity in an adjacent reaction containerof a reaction container in which a fixing reagent is arranged and FIG.25B shows luminescence intensity in a reaction container in which afixing reagent is arranged. The horizontal axis of the graph representsa reaction time and the vertical axis thereof represents fluorescenceintensity. As is evident from these graphs, only the graph of thereaction container in which the fixing reagent is arranged in FIG. 25Bshows that the reaction proceeded without any problems. Moreover, noproceeding reaction is observed in the adjacent reaction container inwhich no fixing reagent is arranged of the reaction container in whichthe fixing reagent is arranged, which shows that the fixing reagent canbe concealed by the sealing compound without causing any problem.

On the other hand, FIGS. 26A and 26B show results of reactions performedin Comparative Example 2, and FIG. 26A shows luminescence intensity inan adjacent reaction container of a reaction container in which a fixingreagent is arranged and FIG. 26B shows luminescence intensity in areaction container in which a fixing reagent is arranged. These resultsshow that the reaction did not proceed at all in the reaction containerin which the fixing reagent is arranged, either. This can be consideredthat because the fixing reagent S is arranged in the recess 27 on thesubstrate 23 side having an aluminum plate, heat during heat sealing ofthe cover material 22 and the substrate 23 is transmitted throughaluminum with a high thermal conductivity to add more heat to the fixingreagent S, resulting in lowering or devitalization of activity of thereagent.

The above demonstrates that, according to the reaction method of thepresent invention, reaction data can be measured accurately withoutcausing lowering or devitalization of activity of a reagent.

1. A reaction method using a reaction chip having a plurality ofreaction containers constituted by a pair of base materials and achannel that mutually communicates with the plurality of reactioncontainers, the method comprising the steps of: arranging a reagentinside a recess of a first base material of the pair of base materials,the recess constituting a part of one of the reaction containers formedin the first base material; sealing the reagent with a hot-melt sealingcompound; producing the reaction chip, having the reaction containers inwhich the reagent is arranged and the channel, by sticking together thefirst base material and a second base material of the pair of basematerials, the second base material constituted by a material whosethermal conductivity is higher than that of the first base material;feeding a reagent solution into the reaction containers through thechannel; and causing a reaction of the reagent and the reagent solutionto proceed while heat is added from a side of the second base material,after the reagent and the reagent solution are brought into contact witheach other by heating the reaction chip from the side of the second basematerial to melt the sealing compound.
 2. The reaction method accordingto claim 1, wherein the first base material and the second base materialare stuck together through thermal welding of a sealant layer providedon at least one side of the first base material and the second basematerial by adding heat from the side of the second base material. 3.The reaction method according to claim 1, wherein the recess in thefirst base material and a recess in the second base material constitutethe one reaction container.
 4. The reaction method according to claim 1,wherein a resin material is used as the first base material and ametallic material is used as the second base material.
 5. The reactionmethod according to claim 1, wherein the sealing compound is constitutedby a material that is soluble in neither the reagent nor the reagentsolution.
 6. The reaction method according to claim 1, wherein the onereaction container is a reaction container for enzyme reaction.